A Paternal Methylation Error in the Congenital Hydrocephalic Texas (H-Tx) Rat Is Partially Rescued with Natural Folate Supplements.

Folate deficiencies, folate imbalance and associated abnormal methylation are associated with birth defects, developmental delays, neurological conditions and diseases. In the hydrocephalic Texas (H-Tx) rat, 10-formyl tetrahydrofolate dehydrogenase (FDH) is reduced or absent from the CSF and the nuclei of cells in the brain and liver and this is correlated with decreased DNA methylation. In the present study, we tested whether impaired folate metabolism or methylation exists in sexually mature, unaffected H-Tx rats, which may explain the propagation of hydrocephalus in their offspring. We compared normal Sprague Dawley (SD, n = 6) rats with untreated H-Tx (uH-Tx, n = 6 and folate-treated H-Tx (TrH-Tx, n = 4). Structural abnormalities were observed in the testis of uH-Tx rats, with decreased methylation, increased demethylation, and cell death, particularly of sperm. FDH and FRα protein expression was increased in uH-Tx males but not in folate-treated males but tissue folate levels were unchanged. 5-Methylcytosine was significantly reduced in untreated and partially restored in treated individuals, while 5-hydroxymethylcytosine was not significantly changed. Similarly, a decrease in DNA-methyltransferase-1 expression in uH-Tx rats was partially reversed with treatment. The data expose a significant germline methylation error in unaffected adult male H-Tx rats from which hydrocephalic offspring are obtained. Reduced methylation in the testis and sperm was partially recovered by treatment with folate supplements leading us to conclude that this neurological disorder may not be completely eradicated by maternal supplementation alone.

Subarachnoid cerebrospinal fluid is essential for normal development of the cerebral cortex.

The central nervous system develops around a fluid filled space which persists in the adult within the ventricles, spinal canal and around the outside of the brain and spinal cord. Ventricular fluid is known to act as a growth medium and stimulator of proliferation and differentiation to neural stem cells but the role of CSF in the subarachnoid space has not been fully investigated except for its role in the recently described "glymphatic" system. Fundamental changes occur in the control and coordination of CNS development upon completion of brain stem and spinal cord development and initiation of cortical development. These include changes in gene expression, changes in fluid and fluid source from neural tube fluid to cerebrospinal fluid (CSF), changes in fluid volume, composition and fluid flow pathway, with exit of high volume CSF into the subarachnoid space and the critical need for fluid drainage. We used a number of experimental approaches to test a predicted critical role for CSF in development of the cerebral cortex in rodents and humans. Data from fetuses affected by spina bifida and/or hydrocephalus are correlated with experimental evidence on proliferation and migration of cortical cells from the germinal epithelium in rodent neural tube defects, as well as embryonic brain slice experiments demonstrating a requirement for CSF to contact both ventricular and pial surfaces of the developing cortex for normal proliferation and migration. We discuss the possibility that complications with the fluid system are likely to underlie developmental disorders affecting the cerebral cortex as well as function and integrity of the cortex throughout life.

Cerebral Folate Metabolism in Post-Mortem Alzheimer's Disease Tissues: A Small Cohort Study.

We investigated the cerebral folate system in post-mortem brains and matched cerebrospinal fluid (CSF) samples from subjects with definite Alzheimer's disease (AD) (n = 21) and neuropathologically normal brains (n = 21) using immunohistochemistry, Western blot and dot blot. In AD the CSF showed a significant decrease in 10-formyl tetrahydrofolate dehydrogenase (FDH), a critical folate binding protein and enzyme in the CSF, as well as in the main folate transporter, folate receptor alpha (FRα) and folate. In tissue, we found a switch in the pathway of folate supply to the cerebral cortex in AD compared to neurologically normal brains. FRα switched from entry through FDH-positive astrocytes in normal, to entry through glial fibrillary acidic protein (GFAP)-positive astrocytes in the AD cortex. Moreover, this switch correlated with an apparent change in metabolic direction to hypermethylation of neurons in AD. Our data suggest that the reduction in FDH in CSF prohibits FRα-folate entry via FDH-positive astrocytes and promotes entry through the GFAP pathway directly to neurons for hypermethylation. This data may explain some of the cognitive decline not attributable to the loss of neurons alone and presents a target for potential treatment.

Neonatal hydrocephalus is a result of a block in folate handling and metabolism involving 10-formyltetrahydrofolate dehydrogenase.

Folate is vital in a range of biological processes and folate deficiency is associated with neurodevelopmental disorders such as neural tube defects and hydrocephalus (HC). 10-formyl-tetrahydrofolate-dehydrogenase (FDH) is a key regulator for folate availability and metabolic interconversion for the supply of 1-carbon groups. In previous studies, we found a deficiency of FDH in CSF associated with the developmental deficit in congenital and neonatal HC. In this study, we therefore aimed to investigate the role of FDH in folate transport and metabolism during the brain development of the congenital hydrocephalic Texas (H-Tx) rat and normal (Sprague-Dawley) rats. We show that at embryonic (E) stage E18 and E20, FDH-positive cells and/or vesicles derived from the cortex can bind methyl-folate similarly to folate receptor alpha, the main folate transporter. Hydrocephalic rats expressed diminished nuclear FDH in both liver and brain at all postnatal (P) ages tested (P5, P15, and P20) together with a parallel increase in hepatic nuclear methyl-folate at P5 and cerebral methylfolate at P15 and P20. A similar relationship was found between FDH and 5-methyl cytosine, the main marker for DNA methylation. The data indicated that FDH binds and transports methylfolate in the brain and that decreased liver and brain nuclear expression of FDH is linked with decreased DNA methylation which could be a key factor in the developmental deficits associated with congenital and neonatal HC. Folate deficiency is associated with neurodevelopmental disorders such as neural tube defects and hydrocephalus. 10-formyl-tetrahydrofolate-dehydrogenase (FDH) is a key regulator for folate availability and metabolic interconversion. We show that FDH binds and transports methylfolate in the brain. Moreover, we found that a deficiency of FDH in the nucleus of brain and liver is linked with decreased DNA methylation which could be a key factor in the developmental deficits associated with congenital and neonatal hydrocephalus cells.

Rat model of fractionated (2 Gy/day) 60 Gy irradiation of the liver: long-term effects.

The liver is considered a radiosensitive organ. However, in rats, high single-dose irradiation (HDI) showed only mild effects. Consequences of fractionated irradiation (FI) in such an animal model have not been studied so far. Rats were exposed to selective liver FI (total dose 60 Gy, 2 Gy/day) or HDI (25 Gy) and were killed three months after the end of irradiation. To study acute effects, HDI-treated rats were additionally killed at several time points between 1 and 48 h. Three months after irradiation, no differences between FI and HDI treatment were found for macroscopically detectable small "scars" on the liver surface and for an increased number of neutrophil granulocytes distributed in the portal fields and through the liver parenchyma. As well, no changes in HE-stained tissues or clear signs of fibrosis were found around the portal vessels. Differences were seen for the number of bile ducts being increased in FI- but not in HDI-treated livers. Serum levels indicative of liver damage were determined for alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (γGT) and lactate dehydrogenase (LDH). A significant increase of AP was detected only after FI while HDI led to the significant increases of AST and LDH serum levels. By performing RT-PCR, we detected up-regulation of matrix metalloproteinases, MMP-2, MMP-9, MMP-14, and of their inhibitors, TIMP-1, TIMP-2 and TIMP-3, shortly after HDI, but not at 3 month after FI or HDI. Overall, we saw punctual differences after FI and HDI, and a diffuse formation of small scars at the liver surface. Lack of "provisional clot"-formation and absence of recruitment of mononuclear phagocytes could be one explanation for scar formation as incomplete repair response to irradiation.

A hybrid deep learning-based intrusion detection system for IoT networks.

The Internet of Things (IoT) is a rapidly evolving technology with a wide range of potential applications, but the security of IoT networks remains a major concern. The existing system needs improvement in detecting intrusions in IoT networks. Several researchers have focused on intrusion detection systems (IDS) that address only one layer of the three-layered IoT architecture, which limits their effectiveness in detecting attacks across the entire network. To address these limitations, this paper proposes an intelligent IDS for IoT networks based on deep learning algorithms. The proposed model consists of a recurrent neural network and gated recurrent units (RNN-GRU), which can classify attacks across the physical, network, and application layers. The proposed model is trained and tested using the ToN-IoT dataset, specifically collected for a three-layered IoT system, and includes new types of attacks compared to other publicly available datasets. The performance analysis of the proposed model was carried out by a number of evaluation metrics such as accuracy, precision, recall, and F1-measure. Two optimization techniques, Adam and Adamax, were applied in the evaluation process of the model, and the Adam performance was found to be optimal. Moreover, the proposed model was compared with various advanced deep learning (DL) and traditional machine learning (ML) techniques. The results show that the proposed system achieves an accuracy of 99% for network flow datasets and 98% for application layer datasets, demonstrating its superiority over previous IDS models.

Ferroportin-1 is a 'nuclear'-negative acute-phase protein in rat liver: a comparison with other iron-transport proteins.

Liver is the central organ of iron metabolism. During acute-phase-response (APR), serum iron concentration rapidly decreases. The current study aimed to compare expression and localization of iron transport protein ferroportin-1 (Fpn-1) and of other iron import proteins after experimental tissue damage induced by injecting turpentine oil in the hind limbs of rats and mice. Serum and spleen iron concentration decreased with an increase in total liver, cytoplasmic and nuclear iron concentration. In liver, mRNA amount of Fpn-1, Fpn-1a, Fpn-1b, HFE, hemojuvelin (HJV) and hephaestin (heph) genes showed a rapid decrease. Hepcidin, divalent metal transporter-1 (DMT-1), transferrin (Tf) and Tf-receptor-1 (TfR1), TfR-2 (TfR2) gene expression was increased. Western blot analysis of liver tissue lysate confirmed the changes observed at mRNA level. In spleen, a rapid decrease in gene expression of Fpn-1, Fpn-1a, Fpn-1b, DMT-1, Tf, TfR1 and TfR2, and an increase in hepcidin was observed. Immunohistochemistry of DMT-1 and TfR2 were mainly detected in the nucleus of rat liver and spleen, whereas TfR1 was clearly localized in the plasma membrane. Fpn-1 was mostly found in the nuclei of liver cells, whereas in spleen, the protein was mainly detected in the cell membrane. Western blot analysis of liver fractions confirmed immunohistochemical results. In livers of wild-type mice, gene expression of Fpn-1, Fpn-1a and Fpn-1b was downregulated, whereas hepcidin gene expression was increased. In contrast, these changes were less pronounced in IL-6ko-mice. Cytokine (IL-6, IL-1b and TNF-a) treatment of rat hepatocytes showed a downregulation of Fpn-1, Fpn-1a and Fpn-1b, and upregulation of hepcidin gene expression. Moreover, western blot analysis of cell lysate of IL-6-treated hepatocytes detected, as expected, an increase of a2-macroglobulin (positive acute-phase protein), whereas albumin (negative acute-phase protein) and Fpn-1 were downregulated. Our results demonstrate that liver behaves as a 'sponge' for iron under acute-phase conditions, and Fpn-1 behaves as a negative acute-phase protein in rat hepatocytes mainly, but not exclusively, because of the effect of IL-6. These changes could explain iron retention in the cytoplasm and in the nucleus of hepatocytes during APR.

Immunodetection of cyclooxygenase-2 (COX-2) is restricted to tissue macrophages in normal rat liver and to recruited mononuclear phagocytes in liver injury and cholangiocarcinoma.

It has been suggested that cyclooxygenase-2 (COX-2)-mediated prostaglandin synthesis is associated with liver inflammation and carcinogenesis. The aim of this study is to identify the cellular source of COX-2 expression in different stages, from acute liver injury through liver fibrosis to cholangiocarcinoma (CC). We induced in rats acute and "chronic" liver injury (thioacetamide (TAA) or carbon tetrachloride (CCl(4))) and CC development (TAA) and assessed COX-2 gene expression in normal and damaged liver tissue by RT-PCR of total RNA. The cellular localization of COX-2 protein in liver tissue was analyzed by immunohistochemistry as well as in isolated rat liver cells by Western blotting. The findings were compared with those obtained in human cirrhotic liver tissue. The specificity of the antibodies was tested by 2-DE Western blot and mass spectrometric identification of the positive protein spots. RT-PCR analysis of total RNA revealed an increase of hepatic COX-2 gene expression in acutely as well as "chronically" damaged liver. COX-2-protein was detected in those ED1(+)/ED2(+) cells located in the non-damaged tissue (resident tissue macrophages). In addition COX-2 positivity in inflammatory mononuclear phagocytes (ED1(+)/ED2(-)), which were also present within the tumoral tissue was detected. COX-2 protein was clearly detectable in isolated Kupffer cells as well as (at lower level) in isolated "inflammatory" macrophages. Similar results were obtained in human cirrhotic liver. COX-2 protein is constitutively detectable in liver tissue macrophages. Inflammatory mononuclear phagocytes contribute to the increase of COX-2 gene expression in acute and chronic liver damage induced by different toxins and in the CC microenvironment.

Folate Related Pathway Gene Analysis Reveals a Novel Metabolic Variant Associated with Alzheimer's Disease with a Change in Metabolic Profile.

Metabolic disorders may be important potential causative pathways to Alzheimer's disease (AD). Cerebrospinal fluid (CSF) decreasing output, raised intracranial pressure, and ventricular enlargement have all been linked to AD. Cerebral folate metabolism may be a key player since this is significantly affected by such changes in CSF, and genetic susceptibilities may exist in this pathway. In the current study, we aimed to identify whether any single nucleotide polymorphism (SNPs) affecting folate and the associated metabolic pathways were significantly associated with AD. We took a functional nutrigenomics approach to look for SNPs in genes for the linked folate, methylation, and biogenic amine neurotransmitter pathways. Changes in metabolism were found with the SNPs identified. An abnormal SNP in methylene tetrahydrofolate dehydrogenase 1 (MTHFD1) was significantly predictive of AD and associated with an increase in tissue glutathione. Individuals without these SNPs had normal levels of glutathione but significantly raised MTHFD1. Both changes would serve to decrease potentially neurotoxic levels of homocysteine. Seven additional genes were associated with Alzheimer's and five with normal ageing. MTHFD1 presents a strong prediction of susceptibility and disease among the SNPs associated with AD. Associated physiological changes present potential biomarkers for identifying at-risk individuals.

Ensemble learning-based IDS for sensors telemetry data in IoT networks.

The Internet of Things (IoT) is a paradigm that connects a range of physical smart devices to provide ubiquitous services to individuals and automate their daily tasks. IoT devices collect data from the surrounding environment and communicate with other devices using different communication protocols such as CoAP, MQTT, DDS, etc. Study shows that these protocols are vulnerable to attack and prove a significant threat to IoT telemetry data. Within a network, IoT devices are interdependent, and the behaviour of one device depends on the data coming from another device. An intruder exploits vulnerabilities of a device's interdependent feature and can alter the telemetry data to indirectly control the behaviour of other dependent devices in a network. Therefore, securing IoT devices have become a significant concern in IoT networks. The research community often proposes intrusion Detection Systems (IDS) using different techniques. One of the most adopted techniques is machine learning (ML) based intrusion detection. This study suggests a stacking-based ensemble model makes IoT devices more intelligent for detecting unusual behaviour in IoT networks. The TON-IoT (2020) dataset is used to assess the effectiveness of the proposed model. The proposed model achieves significant improvements in accuracy and other evaluation measures in binary and multi-class classification scenarios for most of the sensors compared to traditional ML algorithms and other ensemble techniques.

Myeloperoxidase and elastase are only expressed by neutrophils in normal and in inflamed liver.

Myeloperoxidase (MPO) is involved in acute and chronic inflammatory diseases. The source of MPO in acute liver diseases is still a matter of debate. Therefore, we analysed MPO-gene expression on sections from normal and acutely damaged [carbon tetrachloride-(CCl(4)) or whole liver γ-Irradiation] rat liver by immunohistochemistry, real time PCR and Western blot analysis of total RNA and protein. Also total RNA and protein from isolated Kupffer cells, hepatic stellate cells, Hepatocytes, endothelial cells and neutrophil granulocytes (NG) was analysed by real time PCR and Western blot, respectively. Sections of acutely injured human liver were prepared for MPO and CD68 immunofluorescence double staining. In normal rat liver MPO was detected immunohistochemically and by immunofluorescence double staining only in single NG. No MPO was detected in isolated parenchymal and non-parenchymal cell populations of the normal rat liver. In acutely damaged rat liver mRNA of MPO increased 2.8-fold at 24 h after administration of CCl(4) and 3.3-fold at 3 h after γ-Irradiation and MPO was detected by immunofluorescence double staining only in elastase (NE) positive NGs but not in macrophages (ED1 or CD68 positive cells). Our results demonstrate that, increased expression of MPO in damaged rat and human liver is due to recruited elastase positive NGs.

Single-dose gamma-irradiation induces up-regulation of chemokine gene expression and recruitment of granulocytes into the portal area but not into other regions of rat hepatic tissue.

Liver damage is a serious clinical complication of gamma-irradiation. We therefore exposed rats to single-dose gamma-irradiation (25 Gy) that was focused on the liver. Three to six hours after irradiation, an increased number of neutrophils (but not mononuclear phagocytes) was observed by immunohistochemistry to be attached to portal vessels between and around the portal (myo)fibroblasts (smooth muscle actin and Thy-1(+) cells). MCP-1/CCL2 staining was also detected in the portal vessel walls, including some cells of the portal area. CC-chemokine (MCP-1/CCL2 and MCP-3/CCL7) and CXC-chemokine (KC/CXCL1, MIP-2/CXCL2, and LIX/CXCL5) gene expression was significantly induced in total RNA from irradiated livers. In laser capture microdissected samples, an early (1 to 3 hours) up-regulation of CCL2, CXCL1, CXCL8, and CXCR2 gene expression was detected in the portal area but not in the parenchyma; with the exception of CXCL1 gene expression. In addition, treatment with an antibody against MCP-1/CCL2 before irradiation led to an increase in gene expression of interferon-gamma and IP-10/CXCL10 in liver tissue without influencing the recruitment of granulocytes. Indeed, the CCL2, CXCL1, CXCL2, and CXCL5 genes were strongly expressed and further up-regulated in liver (myo)fibroblasts after irradiation (8 Gy). Taken together, these results suggest that gamma-irradiation of the liver induces a transient accumulation of granulocytes within the portal area and that (myo)fibroblasts of the portal vessels may be one of the major sources of the chemokines involved in neutrophil recruitment. Moreover, inhibition of more than one chemokine (eg, CXCL1 and CXCL8) may be necessary to reduce leukocytes recruitment.

Comparison of changes in gene expression of transferrin receptor-1 and other iron-regulatory proteins in rat liver and brain during acute-phase response.

The "acute phase" is clinically characterized by homeostatic alterations such as somnolence, adinamia, fever, muscular weakness, and leukocytosis. Dramatic changes in iron metabolism are observed under acute-phase conditions. Rats were administered turpentine oil (TO) intramuscularly to induce a sterile abscess and killed at various time points. Tissue iron content in the liver and brain increased progressively after TO administration. Immunohistology revealed an abundant expression of transferrin receptor-1 (TfR1) in the membrane and cytoplasm of the liver cells, in contrast to almost only nuclear expression of TfR1 in brain tissue. The expression of TfR1 increased at the protein and RNA levels in both organs. Gene expression of hepcidin, ferritin-H, iron-regulatory protein-1, and heme oxygenase-1 was also upregulated, whereas that of hemojuvelin, ferroportin-1, and the hemochromatosis gene was significantly downregulated at the same time points in both the brain and the liver at the RNA level. However, in contrast to observations in the liver, gene expression of the main acute-phase cytokine (interleukin-6) in the brain was significantly upregulated. In vitro experiments revealed TfR1 membranous protein expression in the liver cells, whereas nuclear and cytoplasmic TfR1 protein was detectable in brain cells. During the non-bacterial acute phase, iron content in the liver and brain increased together with the expression of TfR1. The iron metabolism proteins were regulated in a way similar to that observed in the liver, possibly by locally produced acute-phase cytokines. The significance of the presence of TfR1 in the nucleus of the brain cells has to be clarified.

A Decade of Mighty Lipophagy: What We Know and What Facts We Need to Know?

Lipids are integral cellular components that act as substrates for energy provision, signaling molecules, and essential constituents of biological membranes along with a variety of other biological functions. Despite their significance, lipid accumulation may result in lipotoxicity, impair autophagy, and lysosomal function that may lead to certain diseases and metabolic syndromes like obesity and even cell death. Therefore, these lipids are continuously recycled and redistributed by the process of selective autophagy specifically termed as lipophagy. This selective form of autophagy employs lysosomes for the maintenance of cellular lipid homeostasis. In this review, we have reviewed the current literature about how lipid droplets (LDs) are recruited towards lysosomes, cross-talk between a variety of autophagy receptors present on LD surface and lysosomes, and lipid hydrolysis by lysosomal enzymes. In addition to it, we have tried to answer most of the possible questions related to lipophagy regulation at different levels. Moreover, in the last part of this review, we have discussed some of the pathological states due to the accumulation of these LDs and their possible treatments under the light of currently available findings.

Changes in gene expression of DOR and other thyroid hormone receptors in rat liver during acute-phase response.

Non-thyroidal illness is characterized by low tri-iodothyronine (T3) serum level under acute-phase conditions. We studied hepatic gene expression of the newly identified thyroid hormone receptor (TR) cofactor DOR/TP53INP2 together with TRs in a rat model of aseptic abscesses induced by injecting intramuscular turpentine-oil into each hind limb. A fast (4-6 h) decrease in the serum level of free thyroxine and free T3 was observed. By immunohistology, abundant DOR protein expression was detected in the nuclei of hepatocytes and ED-1(+) (mononuclear phagocytes), CK-19(+) (biliary cells), and SMA(+) (mesenchymal cells of the portal tract) cells. DOR signal was reduced with a minimum at 6-12 h after the acute-phase reaction (APR). Immunohistology also showed a similar pattern of protein expression in TRα1 but without a significant change during APR. Transcripts specific for DOR, nuclear receptor co-repressor 1 (NCoR-1), and TRß1 were down-regulated with a minimum at 6-12 h, whereas expression for TRα1 and TRα2 was slightly and significantly up-regulated, respectively, with a maximum at 24 h after APR was initiated. In cultured hepatocytes, acute-phase cytokines interleukin-1ß (IL-1ß) and IL-6 down-regulated DOR and TRß1 at the mRNA level. Moreover, gene expression of DOR and TRs (TRα1, TRα2, and TRß1) was up-regulated in hepatocytes by adding T3 to the culture medium; this up-regulation was almost completely blocked by treating the cells with IL-6. Thus, TRß1, NCoR-1, and the recently identified DOR/TP53INP2 are abundantly expressed and down-regulated in liver cells during APR. Their down-regulation is attributable to the decreased serum level of thyroid hormones and most probably also to the direct action of the main acute-phase cytokines.

Altered folate binding protein expression and folate delivery are associated with congenital hydrocephalus in the hydrocephalic Texas rat.

Hydrocephalus (HC) is an imbalance in cerebrospinal fluid (CSF) secretion/absorption resulting in fluid accumulation within the brain with consequential pathophysiology. Our research has identified a unique cerebral folate system in which depletion of CSF 10-formyl-tetrahydrofolate-dehydrogenase (FDH) is associated with cortical progenitor cell-cycle arrest in hydrocephalic Texas (H-Tx) rats. We used tissue culture, immunohistochemistry, in-situ PCR and RT-PCR and found that the in-vitro proliferation of arachnoid cells is highly folate-dependent with exacerbated proliferation occurring in hydrocephalic CSF that has low FDH but high folate-receptor-alpha (FRα) and folate. Adding FDH to this CSF prevented aberrant proliferation indicating a regulatory function of FDH on CSF folate concentration. Arachnoid cells have no detectable mRNA for FRα or FDH, but FDH mRNA is found in the choroid plexus (CP) and CSF microvesicles. Co-localization of FDH, FRα and folate suggests important functions of FDH in cerebral folate transport, buffering and function. In conclusion, abnormal CSF levels of FDH, FRα and folate inhibit cortical cell proliferation but allow uncontrolled arachnoid cell division that should increase fluid absorption by increasing the arachnoid although this fails in the hydrocephalic brain. FDH appears to buffer available folate to control arachnoid proliferation and function.

Gene and Cell Therapy for Muscular Dystrophies: Are We Getting There?

In the last few years, significant advances have occurred in the preclinical and clinical work toward gene and cell therapy for muscular dystrophy. At the time of this writing, several trials are ongoing and more are expected to start. It is thus a time of expectation, even though many hurdles remain and it is unclear whether they will be overcome with current strategies or if further improvements will be necessary.

Mediators of hypoxia in a rat model of sterile-induced acute liver injury.

BACKGROUND: The liver plays a key role in iron homeostasis during injury and hypoxia. METHODS: For induction of liver injury, thioacetamide (TAA) was administered intraperitoneally to male Sprague Dawley rats. Animals were sacrificed at 0, 1, 3, 6, 12, 24, 48, 72 and 96 h. Serum, liver, spleen and heart tissues were collected from control and TAA-treated rats. Tissue sections were prepared for immunohistochemical studies. Nuclear and cytoplasmic proteins were isolated for Western blot analysis. RESULTS: Hypoxia inducible factor (HIF)-1α and ED1 positive cells accumulated around the portal field and the interlobular space within 12 hours after TAA administration. Accordingly, Western blot analysis of liver tissue showed an early increase of HIF1α followed by a decrease at 48 h to 96 h. For Erythropoietin (EPO), as well as for HIF1- and -2α, a time-dependent translocation was observed from the cytoplasmic to the nuclear compartment. CONCLUSION: Our data suggest that the TAA-induced acute liver damage generates HIF-1α dependent rescue mechanisms with translocation of EPO from the cytoplasmic to the nuclear compartment. Enhanced iron transport into the liver could be necessary for increased metabolic activities during repair processes.

Reabsorption of iron into acutely damaged rat liver: A role for ferritins.

AIM: To studied iron metabolism in liver, spleen, and serum after acute liver-damage, in relation to surrogate markers for liver-damage and repair. METHODS: Rats received intraperitoneal injection of the hepatotoxin thioacetamide (TAA), and were sacrificed regularly between 1 and 96 h thereafter. Serum levels of transaminases and iron were measured using conventional laboratory assays. Liver tissue was used for conventional histology, immunohistology, and iron staining. The expression of acute-phase cytokines, ferritin light chain (FTL), and ferritin heavy chain (FTH) was investigated in the liver by qRT-PCR. Western blotting was used to investigate FTL and FTH in liver tissue and serum. Liver and spleen tissue was also used to determine iron concentrations. RESULTS: After a short initial decrease, iron serum concentrations increased in parallel with serum transaminase (aspartate aminotransferase and alanine aminotransferase) levels, which reached a maximum at 48 h, and decreased thereafter. Similarly, after 48 h a significant increase in FTL, and after 72h in FTH was detected in serum. While earliest morphological signs of inflammation in liver were visible after 6 h, increased expression of the two acute-phase cytokines IFN-γ (1h) and IL-1ß (3h) was detectable earlier, with maximum values after 12-24 h. Iron concentrations in liver tissue increased steadily between 1 h and 48 h, and remained high at 96 h. In contrast, spleen iron concentrations remained unchanged until 48 h, and increased mildly thereafter (96 h). Although tissue iron staining was negative, hepatic FTL and FTH protein levels were strongly elevated. Our results reveal effects on hepatic iron concentrations after direct liver injury by TAA. The increase of liver iron concentrations may be due to the uptake of a significant proportion of the metal by healthy hepatocytes, and only to a minor extent by macrophages, as spleen iron concentrations do not increase in parallel. The temporary increase of iron, FTH and transaminases in serum is obviously due to their release by damaged hepatocytes. CONCLUSION: Increased liver iron levels may be the consequence of hepatocyte damage. Iron released into serum by damaged hepatocytes is obviously transported back and stored via ferritins.

Regulation of iron uptake in primary culture rat hepatocytes: the role of acute-phase cytokines.

Decreased serum and increased hepatic iron uptake is the hallmark of acute-phase (AP) response. Iron uptake is controlled by iron transport proteins such as transferrin receptors (TfRs) and lipocalin 2 (LCN-2). The current study aimed to understand the regulation of iron uptake in primary culture hepatocytes in the presence/absence of AP mediators. Rat hepatocytes were stimulated with different concentrations of iron alone (0.01, 0.1, 0.5 mM) and AP cytokines (interleukin 6 [IL-6], IL-1ß, tumor necrosis factor α) in the presence/absence of iron (FeCl3: 0.1 mM). Hepatocytes were harvested at different time points (0, 6, 12, 24 h). Total mRNA and proteins were extracted for reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. A significant iron uptake was detected with 0.1 mM iron administration with a maximum (133.37 ± 4.82 µg/g of protein) at 24 h compared with control and other iron concentrations. This uptake was further enhanced in the presence of AP cytokines with a maximum iron uptake (481 ± 25.81 µg/g of protein) after concomitant administration of IL-6 + iron to cultured hepatocytes. Concomitantly, gene expression of LCN-2 and ferritin subunits (light- and heavy-chain ferritin subunits) was upregulated by iron or/and AP cytokines with a maximum at 24 h both at mRNA and protein levels. In contrast, a decreased TfR1 level was detected by IL-6 and iron alone, whereas combination of iron and AP cytokines (mainly IL-6) abrogated the downregulation of TfR1. An increase in LCN-2 release into the supernatant of cultured hepatocytes was observed after addition of iron/AP cytokines into the medium. This increase in secretion was further enhanced by combination of IL-6 + iron. In conclusion, iron uptake is tightly controlled by already present iron concentration in the culture. This uptake can be further enhanced by AP cytokines, mainly by IL-6.